Methods of screening for FabK antagonists and agonists

ABSTRACT

The invention provides methods for identifying agonists and antagonists of FabK polypeptides.

This application claims the benefit of U.S. Provisional Application No.60/238,366, filed Oct. 6, 2000.

FIELD OF THE INVENTION

This invention relates to methods of using polynucleotides andpolypeptides of the Fab family, as well as their variants, hereinreferred to as “FabK,” “FabK polynucleotide(s),” and “FabKpolypeptide(s),” as the case may be.

BACKGROUND OF THE INVENTION

Infections caused by or related to bacteria are a major cause of humanillness worldwide, and the frequency of resistance to standardantibiotics has risen dramatically over the last decade. Hence, thereexists an unmet medical need and demand for new anti-microbial agentsagainst pathogenic bacteria, as well as drug screening methods toidentify such agents.

An example of a bacterial enzyme that is resistant to a widely usedantibacterial agent, Triclosan, is FabK. This enzyme, involved in fattyacid biosynthesis, has been recently reported from Streptococcuspneumoniae, a well-known human pathogen (Heath, et al. Nature 406: 145(2000)). The specific activity of the enzyme under the publishedconditions was 64+/−4 nmol min⁻¹, too low to efficiently screen forcompounds that modulate the activity of the enzyme, such as inhibitors(Heath, et al. Nature 406: 145 (2000)). The present invention solvesthis problem by providing a method for screening for FabK agonists andantagonists, wherein FabK activity is sufficient to perform efficientcompound screening.

A further problem identified by recent studies has been solved by thisinvention. Heath teaches that organisms expressing FabK will berefractory to FabI inhibitors, and that bacteria possessing both targetswill require a combination of inhibitors to block growth (Heath, et al.Nature 406: 145 (2000)). The present invention provides methods ofscreening for compounds that inhibit both enzymes, as well as the use ofsuch compounds as antimicrobial compounds.

SUMMARY OF THE INVENTION

The present invention relates methods using FabK, in particular FabKpolypeptides and FabK polynucleotides, to screen for antimicrobialcompounds.

Also provided by the invention is a method of screening for an agonistor antagonist of FabK polypeptide comprising the steps of: providing areaction mixture comprising a FabK polypeptide; contacting a candidatecompound to said reaction mixture; and detecting activation orinhibition of an activity of said FabK polypeptide.

Another aspect of the invention is a method for screening to identifycompounds that activate or that inhibit an activity of Fab K polypeptidecomprising a method selected from the group consisting of: (a) measuringthe binding of a candidate compound to the polypeptide (or to the cellsor membranes bearing the polypeptide) or a fusion protein thereof bymeans of a label directly or indirectly associated with the candidatecompound; (b) measuring the binding of a candidate compound to thepolypeptide (or to the cells or membranes bearing the polypeptide) or afusion protein thereof in the presence of a labeled competitor; (c)testing whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide, using detection systemsappropriate to the cells or cell membranes bearing the polypeptide; or(d) mixing a candidate conmpound with a solution comprising a FabKpolypeptide, to form a mixture, measuring activity of the polypeptide inthe mixture, and comparing the activity of the mixture to a standard.

A still further aspect of the invention provides a method wherein saidreaction mixture comprises crotonyl ACP and/or an ACP comprising alinked 6 to 8 carbon chain, and/or NADH and/or a cation.

Another aspect of the invention is a method wherein said detecting stepcomprises measuring a change in light absorption at least 2 time points.

The invention also provides a method wherein FabK polypeptide is anisolated polypeptide selected from the group consisting of: (i) anisolated polypeptide comprising an amino acid having at least 95%identity to the amino acid sequence of SEQ ID NO:2 over the entirelength of SEQ ID NO:2; (ii) an isolated polypeptide comprising the aminoacid sequence of SEQ ID NO:2, (iii) an isolated polypeptide that is theamino acid sequence of SEQ ID NO:2, and (iv) a polypeptide that isencoded by a recombinant polynucleotide comprising the polynucleotidesequence of SEQ ID NO:1.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to methods using FabK polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to methods using polypeptides and polynucleotides of aFabK of Streprococcus pneumoniae. The invention relates especially tomethods using FabK having a nucleotide and amino acid sequences set outin Table 1 as SEQ ID NO:1 and SEQ ID NO:2 respectively. The start codonin SEQ ID NO: 1 begins with nucleotide 1 and the stop codon ends withnucleotide 975. Note that sequences recited in the Sequence Listingbelow as “DNA” represent an exemplification of the invention, sincethose of ordinary skill will recognize that such sequences can beusefully employed in. polynucleotides in general, includingribopolynucleotides.

TABLE 1 FabK Polynucleotide and Polypeptide Sequences (A) Streptococcuspneumoniae FabK polynucleotide sequence [SEQ ID NO: 1].5′-ATGAAAACGCGTATTACAGAATTATTGAAGATTGAcTATCCTATTTTCCAAGGAGGGATGGCCTGGGTTGCTGATGGTGATTTGGCAGGGGCTGTTTCCAAGGCTGGAGGATTAGGAATTATCGGTGGGGGAAATGCCCCGAAAGAAGTTGTCAAGGCCAATATTGATAAAATCAAATCATTGACTGATAAACCCTTTGGGGTCAACATCATGCTCTTATCTCCCTTTGTGGAAGAtATCGTGGATCTCGTTATTGAAGAAGGTGTTAAAGTTGTCACAACAGGAGCAGGAAATCCAAGCAAGTATATGGAACGTTTCCATGAAGCTGGGATAATCGTTATTCCTGTTGTTCCTAGTGTCGCTTTAGCTAAACGCATGGAAAAAATCGGTGCAGACGCTGTTATTGCAGAAGGAATGGAAGCTGGGGGGCATATCGGTAAATTAACAACCATGACCTTGGTGCGACAGGTAGCCACAGCTATATCTATTCCTGTTATTGCTGCAGGAGGAATTGCGGATGGTGAAGGTGCTGCGGCTGGCTTTATGCTAGGTGCAGAGGCTGTACAGGTGGGGACACGGTTTGTAGTTGCAAAAGAGTCGAATGCCCATCCAAACTACAAGGAGAAAATTTTAAAAGCAAGGGATATTGATACTACGATTTCAGCTCAGCACTTTGGTCATGCTGTTCGTGCTATTAAAAATCAGTTGACTAGAGATTTTGAACTGGCTGAAAAAGATGCCTTTAAGCAGGAAGATCCTGATTTAGAAATCTTTGAACAAATGGGAGCAGGTGCCCTAGCCAAAGCAGTTGTTCACGGTGATGTGGATGGTGGCTCTGTCATGGCAGGTCAAATCGCAGGGCTTGTTTCTAAAGAAGAAACAGCTGAAGAAATCCTAAAAGATTTGTATTACGGAGCCGCTAAGAAAATTCAAGAAGAAGCCTCTCGCTGGGCAGGAGTTGTAAGAAATGACT AA-3′(B) Streptococcus pneumoniae FabK polypeptide sequence deduced from apolynucleotide sequence in this table [SEQ ID NO:2]. NH₂-MKTRITELLKIDYPIFQGGMAWVADGDLAGAVSKAGGLGIIGGGNAPKEVVKANIDKIKSLTDKPFGVNIMLLSPFVEDIVDLVIEEGVKVVTTGAGNPSKYMERFHEAGIIVIPVVPSVALAKRMEKIGADAVIAEGMEAGGHIGKLTTMTLVRQVATAISIPVIAAGGIADGEGAAAGFMLGAEAVQVGTRFVVAKESNAHPNYKEKILKARDIDTTISAQHFGHAVRAIKNQLTRDFELAEKDAFKQEDPDLEIFEQMGAGALAKAVVHGDVDGGSVMAGQIAGLVSKEETAEEILKDLYYGAAKKIQEEASRWAGVVRND-COOH

Deposited Materials

A deposit comprising a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 IRY,Scotland on 11 Apr. 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit.

On 17 April 1996 a Streptococcus pneumoniae 0100993 DNA library in E.coli was similarly deposited with the NCUMB and assigned deposit number40800. The Streptococcus pneumoniae strain deposit is referred to hereinas “the deposited strain” or as “the DNA of the deposited strain.”

The deposited strain comprises a full length FabK gene. The sequence ofthe polynucleotides comprised in the deposited strain, as well as theamino acid sequence of any polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The deposited strainwill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent. The deposited strain is providedmerely as convenience to those of skill in the art and is not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strain, and compounds derived therefrom, and no suchlicense is hereby granted.

In one aspect of the invention there is provided an isolated nucleicacid molecule encoding a mature polypeptide expressible by theStreptococcus pneumoniae 0100993 strain, which polypeptide is comprisedin the deposited strain. Further provided by the invention are FabKpolynucleotide sequences in the deposited strain, such as DNA and RNA,and amino acid sequences encoded thereby. Also provided by the inventionare FabK polypeptide and polynucleotide sequences isolated from thedeposited strain.

Polypeptides

FabK polypeptide of the invention is substantially phylogeneticallyrelated to other FabK polypeptides from other species.

In one aspect of the invention there are methods provided usingpolypeptides of Streptococcus pneumoniae referred to herein as “FabK”and “FabK polypeptides” as well as biologically, therapeutically orclinically useful variants thereof, and compositions comprising thesame, useful in such methods.

Among the particularly preferred embodiments of the invention aremethods using variants of FabK polypeptide encoded by naturallyoccurring alleles of a FabK gene, particularly in candidate compoundscreening.

The present invention further provides methods using an isolatedpolypeptide that: (a) comprises or consists of an amino acid sequencethat has at least 95% identity, most preferably at least 97-99% or exactidentity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2;(b) a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence that has at least 95% identity,even more preferably at least 97-99% or exact identity to SEQ ID NO:1over the entire length of SEQ ID NO: 1; (c) a polypeptide encoded by anisolated polynucleotide comprising or consisting of a polynucleotidesequence encoding a polypeptide that has at least 95% identity, evenmore preferably at least 97-99% or exact identity, to the amino acidsequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.

The polypeptides of the methods of the invention include, for example, apolypeptide of Table 1 [SEQ ID NO:2] (in particular a maturepolypeptide) as well as polypeptides and fragments, particularly thosethat has a biological activity of FabK, and also those that have atleast 95% identity to a polypeptide of Table 1 [SEQ ID NO:2] and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally comprising at least 30 amino acids and morepreferably at least 50 amino acids, particularly those portionspossessing an activity of a wild type FabK.

The invention also includes methods using a polypeptide consisting of orcomprising a polypeptide of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the amino terminus, X is hydrogen, a metal or any othermoiety described herein for modified polypeptides, and at the carboxylterminus, Y is hydrogen, a metal or any other moiety described hereinfor modified polypeptides, R₁ and R₃ are any amino acid residue ormodified amino acid residue, m is an integer between 1 and 1000 or zero,n is an integer between 1 and 1000 or zero, and R₂ is an amino acidsequence of the invention, particularly an amino acid sequence selectedfrom Table 1 or modified forms thereof. In the formula above, R₂ isoriented so that its amino terminal amino acid residue is at the left,covalently bound to R₁, and its carboxy terminal amino acid residue isat the right, covalently bound to R₃. Any stretch of amino acid residuesdenoted by either R₁ or R₃, where m and/or n is greater than 1, may beeither a heteropolyter or a homopolymer, preferably a heteropolymer.Other preferred embodiments of the invention are provided where m is aninteger between 1 and 50, 100 or 500, and n is an integer between 1 and50, 100, or 500.

It is most preferred in the methods of the invention that thepolypeptide used is derived from Streptococcus pneumoniae; however, itmay also be obtained from other organisms of the same taxonomnic genus.Methods are also provided using polypeptides from organisms of the sametaxonomic family or order, among other organisms as described elsewhereherein.

For the purposes of this invention, a fragment is a variant polypeptidehaving an amino acid sequence that is entirely the same as part but notall of any amino acid sequence of any polypeptide used in the methods ofthe invention. As with FabK polypeptides, fragments may be“free-standing,” or comprised within a larger polypeptide of which theyform a part or region, most preferably as a single continuous region ina single larger polypeptide.

Preferred fragments used in the methods of the invention include, forexample, truncation polypeptides having a portion of an anino acidsequence of Table 1 [SEQ ID NO:2], or of variants thereof, such as acontinuous series of residues that includes an amino- and/orcarboxyl-terminal amino acid sequence, particularly those possessing anenzymatic function of wild type FabK.

Antagonists and Agonists—Assays and Molecules

Polypeptides and polynucleotides described herein may be used to assessthe binding or other effects of small molecule substrates and ligandsin, for example, cells, cell-free preparations, chemical libraries, andnatural product mixtures. These substrates and ligands may be naturalsubstrates and ligands or may be structural or functional mimetics. See,e.g., Coligan et al., Current Protocols in Immunology 1(2): Chapter 5(1991).

Polypeptides and polynucleotides described herein are responsible formany biological functions, including many disease states, in particularthe Diseases herein mentioned, among others. In view of this, thepresent invention provides for a method of screening candidate compoundsto identify those that agonize (e.g., stimulate) or that antagonize(e.g., inhibit) a function of a polypeptide or polynucleotide of theinvention, as well as related polypeptides and polynucleotides. Ingeneral, agonists or antagonists may be employed for therapeutic andprophylactic purposes against such Diseases as herein mentioned.Compounds (herein also “candidate compound(s)”) may be identified orselected from a variety of sources, for example, cells, cell-freepreparations, known or newly synthesized compounds, chemical libraries,and natural product rnixtures. Such compounds, such as agonists andantagonists, so-identified may be natural or modified substrates,ligands, receptors, enzymes, etc., as the case may be, of FabKpolypeptides and polynucleotides; or may be structural or functionalmirnetics thereof (see Coligan et al., Current Protocols in Immunology1(2):Chapter 5 (1991)).

Antagonists of the invention include, among others, small organicmolecules, peptides, polypeptides and antibodies that bind to apolynucleotide and/or polypeptide of the invention and thereby inhibitor extinguish its activity or expression. Antagonists also may be asmall organic molecule, a peptide, a polypeptide, a closely relatedprotein or antibody that binds the same sites on a binding moleculewithout inducing FabK-induced activities. Such antagonists preferablyprevent the action or expression of FabK polypeptides and/orpolynucleotides by excluding FabK polypeptides andlor polynucleotidesfrom binding.

Other antagonists or agonists of the invention include compounds thatalter the binding of a cation to FabK, or alter an activity of a cationon FabK, particularly a monovalent cation. It is preferred that suchantagonists inhibit such binding or lower such activity.

Preferred agonists and antagonists of the invention are those that alsoagonize or antagonize an activity of FabI. An agonist of FabI may act asan antagonist of FabK and visa versa, or such compounds may agonize orantagonize both FabI and FabK. The invention provides that theseagonists and antagonists, when used as antimicrobial compounds, will beless likely to generate resistant mutants than for compounds that onlyact on a single target. This is a significant and surprising discoveryin view of the teachings of Heath indicating that organisms that expressFabK will be refractory to FabI inhibitors, and that bacteria possessingboth targets will require a combination of inhibitors to block growth(Heath, et al. Nature 406: 145 (2000))

Antagonists of the invention further include small molecules that bindto and occupy the binding site of a FabK polypeptide thereby preventingbinding to cellular binding molecules, such that normal biologicalactivity is prevented. Examples of small molecules include but are notlimited to small organic molecules, peptides or peptide-like molecules.Other antagonists include antisense molecules (see Okano, J. Neurochem.56: 560 (1991); OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENEEXPRESSION, CRC Press, Boca Raton, Fla. (1988), for a description ofthese molecules).

Candidate compounds of the invention that are small molecules preferablyhave a molecular weight below 2,000 daltons, more preferably between 300and 1,000 daltons, and most preferably between 400 and 700 daltons. Itis particularly preferred that these small molecules are organicmolecules.

The screening methods of the invention may simply measure the binding ofa candidate compound to a FabK polypeptide or polynucleotide, or tocells or membranes bearing a FabK polypeptide or polynucleotide, or afusion protein of a FabK polypeptide by means of a label directly orindirectly associated with a candidate compound. Alternatively, thescreening method may involve competition with a labeled competitor.Further, these screening methods may test whether a candidate compoundresults in a signal generated by activation or inhibition of a FabKpolypeptide or polynucleotide, using detection systems appropriate tothe cells comprising a FabK polypeptide or polynucleotide. Inhibitors ofactivation are generally assayed in the presence of a known agonist andthe effect on activation by the agonist by the presence of the candidatecompound is observed. Constitutively active polypeptide and/orconstitutively expressed polypeptides and polynucleotides may beemployed in screening methods for inverse agonists, in the absence of anagonist or antagonist, by testing whether the candidate compound resultsin inhibition of activation of the polypeptide or polynucleotide, as thecase may be. Further, the screening methods may simply comprise thesteps of mixing a candidate compound with a solution comprising apolypeptide or polynucleotide of the present invention, to form amixture, measuring FabK polypeptide and/or polynucleotide activity inthe mixture, and comparing the FabK polypeptide and/or polynucleotideactivity of the mixture to a standard. Fusion proteins, such as thosemade from Fc portion and FabK polypeptide, as herein described, can alsobe used for high-throughput screening assays to identify antagonists ofthe polypeptide of the present invention, as well as of phylogeneticallyand and/or functionally related polypeptides (see D. Bennett et al., JMol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentsthat may inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of screening candidate compounds toidentify those that stimulate or inhibit an activity of FabKpolypeptides or polynucleotides, particularly those compounds that arebacteristatic and/or bactericidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagonists, a synthetic reaction mix, a cellular compartrent, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising FabK polypeptidc and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a FabK agonist or antagonist. The ability of acandidate molecule to agonize or antagonize the FabK polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of FabK polypeptide aremost likely to be good antagonists. Molecules that bind well and, as thecase may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocalorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in FabK polynucleotide or polypeptideactivity, and binding assays known in the art.

Polypeptides of the invention may be used to identify membrane bound orsoluble receptors, if any, for such polypeptide, through standardreceptor binding techniques known in the art. These techniques include,but are not limited to, ligand binding and crosslinking assays in whichthe polypeptide is labeled with a radioactive isotope (for instance,¹²⁵I), chemically modified (for instance, biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (e.g., cells, cell membranes,cell supernatants, tissue extracts, bodily materials). Other methodsinclude biophysical techniques such as surface plasmon resonance andspectroscopy. These screening methods may also be used to identifyagonists and antagonists of the polypeptide that compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tumbling rate. Proteincomplexes, such as formed by FabK polypeptide associating with acandidate compound, labeled to comprise a fluorescently-labeled moleculewill have higher polarization values than a fluorescently labeled FabKprotein not bound to a candidate compound. It is preferred that thismethod be used to characterize small molecules that bind FabK.

Surface plasmon resonance can be used to monitor the effect of smallmolecules on FabK polypeptide self-association as well as an associationof FabK polypeptide and another polypeptide or small molecule. FabKpolypeptide can be coupled to a sensor chip at low site density suchthat covalently bound molecules will be monomeric. Solution protein canthen passed over the FabK polypeptide-coated surface and specificbinding can be detected in real-time by monitoring the change inresonance angle caused by a change in local refractive index. Thistechnique can be used to characterize the effect of small molecules onkinetic rates and equilibrium binding constants for FabK polypeptideself-association as well as an association of FabK polypeptide andanother polypeptide or small molecule.

In other embodiments of the invention there are provided methods foridentifying compounds that bind to or otherwise interact with andinhibit or activate an activity or expression of a FabK polypeptideand/or polynucleotide of the invention comprising: contacting a FabKpolypeptide and/or polynucleotide of the invention with a compound to bescreened under conditions to permit binding to or other interactionbetween the compound and the FabK polypeptide and/or polynucleotide toassess the binding to or other interaction with the compound, suchbinding or interaction preferably being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide and/or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity or expression ofthe FabK polypeptide and/or polynucleotide by detecting the presence orabsence of a signal generated from the binding or interaction of thecompound with the FabK polypeptide and/or polynucleotide.

Another example of an assay for FabK agonists or antagonists is acompetitive assay that combines FabK and a potential agonist orantagonist with FabK-binding molecules, recombinant FabK bindingmolecules, natural substrates or ligands, or substrate or ligandmimetics, under appropriate conditions for a competitive inhibitionassay. FabK can be labeled, such as by radioactivity or a colorimetriccompound, such that the number of FabK molecules bound to a bindingmolecule or converted to product can be determined accurately to assessthe effectiveness of the potential agonist antagonist.

A further assay is provided whereby compounds that are agonists orantagonists of FabK are assayed to determine whether they are alsoagonists or antagonists of FabI, and visa versa. Embodiments are alsoprovided where an assay mixture comprises both FabI and FabK.

FabK catalyses the reduction of enoyl-ACPs with the concomitantoxidation of NADH. Screening methods based on this reaction arepreferred embodiments herein. Such methods may comprise the step ofdetecting reduction of crotonoyl-ACP to butyryl-ACP. Detection may bemonitored, for example, by following the change in absorbance at aparticular wavelength in the light spectrum, preferably at 340 nm, or byany other means of assaying NADH oxidation. Assays may be carried out,for example, in Costar 3696 half-area plates, preferably at a finalassay volume of 150 ul on a Spectramax platereader. Preferred substratesused in the methods of the invention are NADH, NADPH, an NADH analogue,crotonoyl ACP, crotonyl CoA, and ACP comprising crotonyl analogues orhomologues, such as compounds having longer carbon chains, particularlyfrom 6 to 8 carbons, among others. Further provided are preferredmethods comprising the step of incubating substrates with FabK enzyme in100 mM N-[2-acetamido]-2 iminodiacetic acid (ADA), pH 6.5, 100 mM NH₄Cl,4% glycerol at 30° C., followed by an incubation step for 30 minutesafter the addition of FabK. This reaction may be monitored at 340 nm,among other wavelengths.

A particularly preferred embodiment is a method comprising the step ofproviding monovalent cations to an assay mnixture. This step enhancesthe activity of FabK enzyre. Any cation, or any compound that increasesor enhances the activity of the enzyme, may be used in this method;however, NH₄ ⁺ is preferred, and a concentration of 100 mM ismost-preferred. Adding cations to the assay mixture is vastly superiorto assaying in the absence of added cation. The activation in thereaction may be about 300-fold over the reaction with no monovalentcations added, such as in a cell-lysate assay without additionalcations.

Using the assay methods of the invention, compounds may be tested forinhibition of FabK. It is preferred that the assay is performed using anassay volume of between 5 and 200 ul of candidate compound, morepreferably 10 and 50 ul of candidate compound, and most preferably 30ul, Preferred methods also utilize NADH, crotonoyl ACP and crotonyl CoAin the assay mixture. In preferred embodiments of the methods of theinvention, the final concentration in the assay mixture is between 10 uMand 50 uM crotonoyl ACP, but is most preferably 25 uM crotonoyl ACP; isbetween 20 uM and 100 uM NADH, but is most preferably 50 uM NADH; and isbetween 0.25 nM and 1.75 nM FabK enzyme, but is most preferably 1.25 nMFabK enzyme.

It is preferred that the assay mixtures are incubated at between 20° C.and 40° C., preferably between 28° C. to 37° C., and most preferably atabout 30° C. Preferred incubation times range between 30 seconds and 1hour, are more preferably between 3 minutes and 30 minutes, and are mostpreferably about 4 or 5 minutes.

In certain preferred embodiments positive controls, without addedcandidate compound, are used in a reaction well to gauge the degree ofagonism or antagonism of a candidate compound. Negative controls,without enzyme, may also be used.

Glossary

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Disease(s)” means any disease caused by or related to infection by abacteria, including, for example, otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994, Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computerprogramns. Computer program methods to determine identity between twosequences include, but are not limited to, the GCG program package(Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1 984)),BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI andother sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410(1990). The well known Smith Waterman algorithm may also be used todetermine identity.

Parameters for polypeptide sequence comparison include the following:Algorithm: Needleman and Wunsch, 1: Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a95, 97 or 100% identity to the reference sequence of SEQ ID NO:1,wherein said polynucleotide sequence may be identical to the referencesequence of SEQ ID NO:1 or may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in SEQ ID NO:1 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of nucleotides in SEQ ID NO:1, or:

n _(n) ≦x _(n)·(x _(n·y)),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO:2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 95, 97 or 100% identity to apolypeptide reference sequence of SEQ ID NO:2, wherein said polypeptidesequence may be identical to the reference sequence of SEQ ID NO:2 ormay include up to a certain integer number of amino acid alterations ascompared to the reference sequence, wherein said alterations areselected fromthe group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of amino acid alterations is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of amino acids in SEQ ID NO:2, or:

n _(a) ≦x _(a)·(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein aiyy non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Organism(s)” means a (i) prokaryote, including but not limited to, amember of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxelia, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillur,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chiamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium.Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israeli, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophiius ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flewneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridiun tetani, Clostridium boiulinum, Treponema pallidum Rickettsiarickettsii, Helicobacter pylori and Chiamydia trachomitis, (ii) anarchaeon, including but not limited to Archaebacter, and (iii) aunicellular or filamentous eukaryote, including but not limited to, aprotozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces,or Candida, and a member of the species Saccharomyces ceriviseae,Kluveromyces lactis, or Candida albicans.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, that may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- and strandedregions or single-, double- and triple-siranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions nay be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that comprise he one or more modified bases. Thus,DNAs or RNAs with backbones modified for stability or for other reasonsare “polynucleotide(s)” as that term is intended herein. Moreover, DNAsor RNAs comprising unusual bases, such as inosine, or modified bases,such as tritylated bases, to name just two examples, are polynucleotidesas the terrn is used herein. It will be appreciated that a great varietyof modifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may comprise amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may comprise many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl tenini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachrnent of flavin, covalentattachment of a herne moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation ofpyroglutarnate, formylation, gannn-carboxylationI; GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutarnic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslationaf Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Recombinant expression system(s)” refers to expression systems orportions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and Ble; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gin; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

The examples below are carried out using standard techniques, that arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Cloning of Streptococcus pneumoniae FabK

The S.pneumoniae fabK, enoyl-ACP reductase gene was PCR amplified fromS.pneumoniae strain 0100993. The forward and reverse primer sequenceswere

5′AGGTTGGAGGCCATATGAAAACGCGTATT3′ (SEQ ID NO:3) and

5′GGCGGATCCTTAGTCATTTCTTACAACTC3′ (SEQ ID NO:4), respectively. An Ndelsite was integrated into the forward primer and a BamHl site into thereverse primer for cloning into pET24b(+). The PCR product was digestedwith the restriction endonucleases Ndel and BamHl and then ligated intopET24b(+), (also digested with Ndel and BamHl). The resulting plasmidwas transformed into sub-cloning efficiency DH5-alpha cells. Thesequence of the pET24bSpfabK expression construct was confirmed by DNAsequencing and the plasmid was transformed into electrocompetent BL21(DE3) cells harboring the tRNA vector pRR692.

Intact FabK is expressed as 25% total cell protein of which 80% issoluble when induced with 0.1 mM IPTG at 37° C. for three hours.

Example 2 Purification of S.pneumoniae FabK

One liter of cells containing the FabK expression construct were grownto an OD600 of 0.6. Expression was induced with 0.1 mM IPTG and thecells were grown for a further 3 h and then harvested. The cell pelletwas resuspended in 10 ml 50 mM Tris pH7.5, 1 mM PMSF, 1 mM Benzatidine,1 mM DTT (buffer A) and lysed by sonication. Cell debris was removed bycentrifugation. The supernatant was loaded onto a Hi-load Q (16/10)column (Pharmacia) equilibrated in buffer A. Protein was eluted over a200 ml gradient of 0-100% buffer B, where buffer B is buffer A+1 M KCl.Fractions containing FabK wereidentified by their absorbance at A460 andby their FabK activity and pooled.

1.5 M ammonium sulphate was added to the pooled fractions and these werethen loaded onto a Hi-load Phenyl sepharose (16/10) column (Pharmacia)equilibrated in 50 mM Tris pH 7.5, 1 mM PMSF, 1 mM Benzamidine, 1 mMDTT, 1.5 M ammonium sulphate. Proteins were eluted with a gradient ofammonium sulphate (1.5 to 0 M) over 200 ml. Fractions containing FabKwere identified as above and pooled. The pooled fractions were bufferexchanged into 100 mM Tris, pH 7.5, 2 mM DTT and glycerol was then addedto 50%. The protein was stored at −20° C. It is preferred that theenzyme be stored with NH₄Cl, which has been found to stabilize theenzyme.

The amino acid sequence of FabK in the Examples is:

MKTRITELLKIDYPIFQGGMAWVADGDLAGAVSKAGOLGIIGGGNAPKEVVKANIDKIKSLTDKPFGVNIMLLSPFVEDIVDLVIEEGVKVVTTGAGNPSKYMERFHEAGIIVUVVPSVALAKRMEKIGADAVIAEGMEAGGHIGKLTTMTLVRQVATAISIPVIAAGGIADGEGAAAGFMLGAEAVQVGTRFVVAKESNAHPNYKEKILKARDIDTTISAQHFGHAVRAIKNQLTRDFELAEKDAFKQEDPDLEBFEQMGAGALAKAVVHGDVDGGSVMAGQIAGLVSKEETAEEILKDLYYGAAKKIQEEASRWAGVVRND (SEQ ID NO:2)

Example 3 FabK Characterization

The identity of the protein was confirmed by N-terminal sequencing andMALDI mass spectrometry. The optical spectrum of the protein wascharacteristic of flavoproteins, showing an absorbance in the 450nmregion. The FAD cofactor was removed by denaturation of the protein andquantified. The ratio of FAD:protein was shown to be approximately 1:1.

Example 4 Assaying the Activity of FabK

FabK catalyses the reduction of enoyl-ACPs with the concommitantoxidation of NADH. Crotonoyl-ACP can be prepared as described below. Thereduction of crotonoyl-ACP to butyryl-ACP can be monitored by followingthe change in absorbance at 340 nm as NADH is oxidised.

Assays were carried out in Costar 3696 half-area plates in a final assayvolume of 150 ul on a Spectramax platereader. Substrates, NADH andcrotonoyl ACP, were incubated with FabK enzyme in 100 mMN-[2-acetamido]-2 iminodiacetic acid (ADA), pH 6.5, 100 mM NH₄Cl, 4%glycerol at 30° C. and the reaction monitored at 340 nm. This assayingcan also be performed using crotonyl CoA, NADPH or an NADH analogue as asubstrate.

Example 5 Activation by Monovalent Cations

FabK was found to be activated by monovalent cations. The greatestactivation was found to be with NH₄ ⁺ at 100 mM, which activated thereaction about 300 fold over the reaction with no monovalent cations.

Example 6 Compound Screening

Using the above assay, compounds can be tested for inhibition of FabK.30 ul of a candidate compounds is added to a well of the plate. 30 ul ofa 250 uM stock of NADH is then added to the well. 60 ul of a 67.5 uMstock of Crotonoyl ACP is added to the well. The plate is incubated at30° C. for 5 min. 30 ul of a 6.25 nM stock of enzyme is then added tothe well (also preincubated at 30° C.) to initiate the reaction. Theplate is then monitored at A340 nm for 30 min at 30° C. Positivecontrols are reactions without compound. Negative controls are reactionswithout enzyme and without compound. Final concentrations in the assaymixture are 25 uM crotonoyl ACP, 50 uM NADH, 1.25 nM enzyme.

Example 7 Synthesis of Crotonoyl-ACP

Crotonoyl-ACP was synthesised using S. pneumoniae ACP synthase tocatalyse the addition of a crotonoyl group from crotonoyl CoA to E.coliapo-acyl carrier protein (ACP).

To a reaction vessel containing 500 mg (58 μmol) of E. coli apo-ACP in20 mM Bis-Tris, pH 6.8, 5 mM MgCl₂, was added 76 mg (81 μmoles) ofcrotonoyl-CoA and 5 mg of S. pneunioniae ACP synthase. The final volumeand pH were adjusted to 100 mL and 6.8, respectively. The pH of thereaction was maintained at 6.8 with NaOH and monitored for completion bymass spectrometry. Conversion was complete within 150 min with nodetectable by-products. The reaction mixture was loaded at 10 mLnlminonto a Q-Sepharose FF column (5×16 cm) pre-equilibrated with 20 mMBis-Tris, pH 6.8. Crotonoyl-ACP was eluted over 2200 ml using a0.2M-0.6M NaCl gradient at a flow rate of 20 ml/min. Fractions weremonitored by mass spectrometry for identity and purity. The appropriatefractions were pooled and concentrated using a YM-3 membrane.

Example 8 FabI Assay Method

FabI enzyme, and methods of making and using it, is disclosed in patentapplications numbered PCT/US00/12104 and EP1997000306506.

FabI catalyses the reduction of enoyl-ACPs with the concommitantoxidation of NAD(P)H. Crotonoyl-ACP can be prepared as described inpatent applications numbered PCT/US00/12104 and EP1997000306506. Thereduction of enoyl-ACPs can be monitored by following the change inabsorbance at 340 nm as NADH is oxidised. Enoyl ACPs (e.g.crotonoyl-ACP) can be replaced by enoyl-CoAs (e.g. crotonoyl-CoA)

Assays were carried out in Costar 3696 half-area plates in a final assayvolume of 150 μl on a Spectramax platereader. Substrates, NADH andcrotonoyl ACP, were incubated with FabI enzyme in 100 mMN-[2-acetamido]-2 iminodiacetic acid (ADA), pH 6.5, 4% glycerol at 30°C. and the reaction monitored at 340 nm. This assaying can also beperformed using crotonyl CoA, NADPH or an NADH analogue as a substrate,or using a substrate suitable for FabK, such as those described above.

Using the above assay, compounds can be tested for inhibition of FabI.30 μl of a candidate compounds is added to a well of the plate. 30 μl ofa 250 μM stock of NADH is then added to the well. 60 μl of a 67.5 μMstock of Crotonoyl ACP is added to the well. The plate is incubated at30° C. for 5 minutes. 30 μl of a 6.25 nM stock of enzyme is then addedto the well (also preincubated at 30° C.) to initiate the reaction. Theplate is then monitored at A340nm for 30 minutes at 30° C. Positivecontrols are reactions without compound. Negative controls are reactionswithout enzyme and without compound. Final concentrations in the assaymixture are 25 μM crotonoyl ACP and 50 μM NADH.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

4 1 975 DNA Streptococcus pneumoniae 1 atgaaaacgc gtattacaga attattgaagattgactatc ctattttcca aggagggatg 60 gcctgggttg ctgatggtga tttggcaggggctgtttcca aggctggagg attaggaatt 120 atcggtgggg gaaatgcccc gaaagaagttgtcaaggcca atattgataa aatcaaatca 180 ttgactgata aaccctttgg ggtcaacatcatgctcttat ctccctttgt ggaagatatc 240 gtggatctcg ttattgaaga aggtgttaaagttgtcacaa caggagcagg aaatccaagc 300 aagtatatgg aacgtttcca tgaagctgggataatcgtta ttcctgttgt tcctagtgtc 360 gctttagcta aacgcatgga aaaaatcggtgcagacgctg ttattgcaga aggaatggaa 420 gctggggggc atatcggtaa attaacaaccatgaccttgg tgcgacaggt agccacagct 480 atatctattc ctgttattgc tgcaggaggaattgcggatg gtgaaggtgc tgcggctggc 540 tttatgctag gtgcagaggc tgtacaggtggggacacggt ttgtagttgc aaaagagtcg 600 aatgcccatc caaactacaa ggagaaaattttaaaagcaa gggatattga tactacgatt 660 tcagctcagc actttggtca tgctgttcgtgctattaaaa atcagttgac tagagatttt 720 gaactggctg aaaaagatgc ctttaagcaggaagatcctg atttagaaat ctttgaacaa 780 atgggagcag gtgccctagc caaagcagttgttcacggtg atgtggatgg tggctctgtc 840 atggcaggtc aaatcgcagg gcttgtttctaaagaagaaa cagctgaaga aatcctaaaa 900 gatttgtatt acggagccgc taagaaaattcaagaagaag cctctcgctg ggcaggagtt 960 gtaagaaatg actaa 975 2 324 PRTStreptococcus pneumoniae 2 Met Lys Thr Arg Ile Thr Glu Leu Leu Lys IleAsp Tyr Pro Ile Phe 1 5 10 15 Gln Gly Gly Met Ala Trp Val Ala Asp GlyAsp Leu Ala Gly Ala Val 20 25 30 Ser Lys Ala Gly Gly Leu Gly Ile Ile GlyGly Gly Asn Ala Pro Lys 35 40 45 Glu Val Val Lys Ala Asn Ile Asp Lys IleLys Ser Leu Thr Asp Lys 50 55 60 Pro Phe Gly Val Asn Ile Met Leu Leu SerPro Phe Val Glu Asp Ile 65 70 75 80 Val Asp Leu Val Ile Glu Glu Gly ValLys Val Val Thr Thr Gly Ala 85 90 95 Gly Asn Pro Ser Lys Tyr Met Glu ArgPhe His Glu Ala Gly Ile Ile 100 105 110 Val Ile Pro Val Val Pro Ser ValAla Leu Ala Lys Arg Met Glu Lys 115 120 125 Ile Gly Ala Asp Ala Val IleAla Glu Gly Met Glu Ala Gly Gly His 130 135 140 Ile Gly Lys Leu Thr ThrMet Thr Leu Val Arg Gln Val Ala Thr Ala 145 150 155 160 Ile Ser Ile ProVal Ile Ala Ala Gly Gly Ile Ala Asp Gly Glu Gly 165 170 175 Ala Ala AlaGly Phe Met Leu Gly Ala Glu Ala Val Gln Val Gly Thr 180 185 190 Arg PheVal Val Ala Lys Glu Ser Asn Ala His Pro Asn Tyr Lys Glu 195 200 205 LysIle Leu Lys Ala Arg Asp Ile Asp Thr Thr Ile Ser Ala Gln His 210 215 220Phe Gly His Ala Val Arg Ala Ile Lys Asn Gln Leu Thr Arg Asp Phe 225 230235 240 Glu Leu Ala Glu Lys Asp Ala Phe Lys Gln Glu Asp Pro Asp Leu Glu245 250 255 Ile Phe Glu Gln Met Gly Ala Gly Ala Leu Ala Lys Ala Val ValHis 260 265 270 Gly Asp Val Asp Gly Gly Ser Val Met Ala Gly Gln Ile AlaGly Leu 275 280 285 Val Ser Lys Glu Glu Thr Ala Glu Glu Ile Leu Lys AspLeu Tyr Tyr 290 295 300 Gly Ala Ala Lys Lys Ile Gln Glu Glu Ala Ser ArgTrp Ala Gly Val 305 310 315 320 Val Arg Asn Asp 3 29 DNA Streptococcuspneumoniae 3 aggttggagg ccatatgaaa acgcgtatt 29 4 29 DNA Streptococcuspneumoniae 4 ggcggatcct tagtcatttc ttacaactc 29

What is claimed is:
 1. A method of screening for an agonist orantagonist of a FabK polypeptide comprising: contacting an isolated FabKpolypeptide comprises SEQ ID NO: 2 with a candidate compound in thepresence of an NH₄ cation; and detecting activation or inhibition of anactivity of said FabK polypeptide wherein said FabK polypeptidecomprises SEQ ID NO: 2 and has enhanced activity in the presence of NH₄⁺.
 2. The method of claim 1 wherein said contacting is carried out inthe presence of enoyl-ACP.
 3. The method of claim 2 wherein saidcontacting is carried out in the presence of NADH.
 4. The method ofclaims 1 wherein said detecting step comprises measuring a change inlight absorption at least 2 time points.
 5. The method of claim 1wherein FabK polypeptide is an isolated polypeptide that is encoded by arecombinant polynucleotide comprising the polynucleotide sequence of SEQID NO:1.
 6. The method of claim 1 wherein said contacting is carried outin the presence of NADPH.
 7. The method of claim 1, wherein saiddetecting step comprises measuring the rate of reduction of enoyl-ACP.8. The method of claim 7, wherein the enoyl-ACP is crotonoyl-ACP.
 9. Themethod of claim 1, wherein said detecting step comprises assaying theconcentration of NADH.
 10. The method of claim 9, wherein the step ofassaying the concentration of NADH comprises measuring absorbance atabout 340 nm.
 11. The method of claim 2, wherein the enoyl-ACP iscrotonoyl-ACP.
 12. The method of claim 1, wherein said NH₄ cation is ata concentration of at least about 100 mM.